A cardiac mitochondrial cAMP signaling pathway regulates calcium accumulation, permeability transition and cell death.

Although cardiac cytosolic cyclic 3',5'-adenosine monophosphate (cAMP) regulates multiple processes, such as beating, contractility, metabolism and apoptosis, little is known yet on the role of this second messenger within cardiac mitochondria. Using cellular and subcellular approaches, we demonstrate here the local expression of several actors of cAMP signaling within cardiac mitochondria, namely a truncated form of soluble AC (sACt) and the exchange protein directly activated by cAMP 1 (Epac1), and show a protective role for sACt against cell death, apoptosis as well as necrosis in primary cardiomyocytes. Upon stimulation with bicarbonate (HCO3(-)) and Ca(2+), sACt produces cAMP, which in turn stimulates oxygen consumption, increases the mitochondrial membrane potential (ΔΨm) and ATP production. cAMP is rate limiting for matrix Ca(2+) entry via Epac1 and the mitochondrial calcium uniporter and, as a consequence, prevents mitochondrial permeability transition (MPT). The mitochondrial cAMP effects involve neither protein kinase A, Epac2 nor the mitochondrial Na(+)/Ca(2+) exchanger. In addition, in mitochondria isolated from failing rat hearts, stimulation of the mitochondrial cAMP pathway by HCO3(-) rescued the sensitization of mitochondria to Ca(2+)-induced MPT. Thus, our study identifies a link between mitochondrial cAMP, mitochondrial metabolism and cell death in the heart, which is independent of cytosolic cAMP signaling. Our results might have implications for therapeutic prevention of cell death in cardiac pathologies.

Possible common central pathway for resistin and insulin in regulating food intake.

AIM: Adipose tissue has been the object of intense research in the field of obesity and diabetes diseases in the last decade. Examination of adipocyte-secreted peptides led to the identification of a unique polypeptide, resistin (RSTN), which has been suggested as a link between obesity and diabetes. RSTN plays a clearly documented role in blocking insulin (INS)-induced hypoglycaemia. As brain injection of INS affects feeding behaviour, we studied the possible interaction between INS and RSTN in food-deprived rats, measuring effects on food intake. In addition, we examined how RSTN might affect neuropeptide Y (NPY)-induced feeding, as studies have shown that rat RSTN can interfere with the NPY system. METHODS: Overnight food-deprived rats were injected into the third brain ventricle (3V) with either INS (10 or 20 mUI), RSTN (0.1-0.4 nmol/rat), or saline before access to food. Another group of rats was injected into the 3V with RSTN alone, NPY alone or RSTN plus NPY. Their food intake and body weight were measured. RESULTS: Our results confirm the hypophagic effect of RSTN on food deprivation-induced food intake, and more importantly, show that RSTN neither potentiates nor blocks the effects of INS on food intake, but does reduce the hyperphagic effect of NPY. CONCLUSION: The observation that RSTN does not modify feeding INS-induced hypophagia, but does influence NPY-induced feeding, points to the possibility that RSTN may be involved in control of food intake through an NPY-ergic mechanism as INS.

NMR and cDNA array analysis prior to heart failure reveals an increase of unsaturated lipids, a glutamine/glutamate ratio decrease and a specific transcriptome adaptation in obese rat heart.

Obesity is a risk factor for heart failure through a set of hemodynamic and hormonal adaptations, but its contribution at the molecular level is not clearly known. Therefore, we investigated the kinetic cardiac transcriptome and metabolome in the Spontaneous Hypertensive Heart Failure (SHHF) rat. The SHHF rat is devoid of leptin signaling when homozygous for a mutation of the leptin receptor (ObR) gene. The ObR-/- SHHF rat is obese at 4 months of age and prone to heart failure after 14 months whereas its lean counterpart ObR-/+ is prone to heart failure after 16 months. We used a set of rat pangenomic high-density macroarrays to monitor left ventricle cardiac transcriptome regulation in 4- and 10-month-old, lean and obese animals. Comparative analysis of left ventricle of 4- and 10-month-old lean rat revealed 222 differentially expressed genes while 4- and 10-month-old obese rats showed 293 differentially expressed genes. (1)H NMR analysis of the metabolome of left ventricular extracts displayed a global decrease of metabolites, except for taurine, and lipid concentration. This may be attributed to gene expression regulation and likely increased extracellular mass. The glutamine to glutamate ratio was significantly lower in the obese group. The relative unsaturation of lipids increased in the obese heart; in particular, omega-3 lipid concentration was higher in the 10-month-old obese heart. Overall, several specific kinetic molecular patterns act as a prelude to heart failure in the leptin signaling deficient SHHF obese rat.

Insulin downregulates M(2)-muscarinic receptors in adult rat atrial cardiomyocytes: a link between obesity and cardiovascular complications.

OBJECTIVE: To determine whether decreased cardiac parasympathetic activity observed in obesity is due to insulin-induced alterations in cardiac M(2)-muscarinic receptors and/or adenylyl cyclase activity. DESIGN AND METHODS: After incubation with increasing concentrations of insulin, adult rat atrial cardiomyocytes were assayed for M(2)-muscarinic receptor binding density and affinity, and for M(2)R mRNA expression using RT-PCR analysis. Forskolin-stimulated adenylyl cyclase activity and its inhibition by carbachol were also assayed, as was endothelial nitric oxide synthase mRNA expression. The effects of insulin on M(2)-muscarinic receptor density and mRNA expression levels were analyzed using the insulin signaling inhibitors rapamycin, wortmanin and PD 098059. RESULTS: Insulin induces a concentration- and time-dependent decrease in expression of the M(2)R mRNA, and in [(3)H]N-methylscopolamine binding by the receptor. These effects on the M(2)R mRNA levels and on [(3)H]N-methylscopolamine binding were prevented by PD 98059, but not by wortmanin or rapamycin. Basal and forskolin-induced cAMP production did not differ, but the inhibition of forskolin-simulated enzyme activity by carbachol was blunted by insulin. No change in the mRNA levels for endothelial nitric oxide synthase was observed. CONCLUSION: In rat atrial cardiomyocytes, insulin markedly alters both the M(2)-muscarinic receptor density, and its mRNA expression through transcriptional regulation and adenylyl cyclase activity. These data suggest that the obesity-associated decrease in cardiac parasympathetic tone may be related to hyperinsulinemia, which could directly contribute to cardiovascular morbidity in obese patients.

Characterization of semicarbazide-sensitive amine oxidase in human subcutaneous adipocytes and search for novel functions.

Numerous studies have characterized semicarbazide-sensitive amine oxidase activity (SSAO) in rat fat cells but this oxidase is scarcely documented in human adipose tissue. Our aim was to further characterize SSAO in human adipose tissue (activity, mRNA and protein abundance) and to investigate whether SSAO activity can interplay with glucose and lipid metabolism in human adipocytes via the hydrogen peroxide it generates. Polyclonal antibodies directed against bovine lung SSAO allowed the detection of a substantial amount of immunoreactive protein (apparent molecular mass 100 kDa) in human subcutaneous adipocytes from either mammary or abdominal fat depots. A 4-kb mRNA was detected in fat depots using a cDNA probe designed from the placenta SSAO sequence. Almost all the oxidation of benzylamine found in adipose tissue homogenates was due to fat cells and was located in the adipocyte membrane fraction. The oxidation of benzylamine and methylamine were similar and totally inhibited by semicarbazide or hydralazine but resistant to pargyline. Histamine was poorly oxidized. Benzylamine and methylamine dose-dependently stimulated glucose transport in intact adipocytes. This insulin-like effect of amines did not increase in the presence of 0.1 mM vanadate but was inhibited by semicarbazide and antioxidants. Benzylamine and methylamine also exhibited antilipolytic effects, with complete inhibition of lipolysis at 1 mM. These results show that fat cells from non-obese subjects express a membrane-bound SSAO which readily oxidizes exogenous amines, generates hydrogen peroxide and exerts short-term insulin-like actions on glucose and lipid metabolism.

[Study of ventricular repolarization in an experimental model of arterial hypertension associated with obesity]. / Etude de la repolarisation ventriculaire dans un modèle expérimental d'HTA associée à l'obésité.

UNLABELLED: High fat diet (HFD) in dogs is associated with obesity and hypertension (HTN) but also with a significant and early decrease in heart rate variability (HRV). Decreased HRV has been shown to be a good predictor of sudden cardiac death due mainly to arrhythmic event. The aim of this work was to investigate the changes in ventricular repolarization through 24 hours EKG recordings in dogs with hypertension and rendered obese by 20 weeks of HFD. This was achieved through 24 hour EKG recording analysis of QT parameters. The aims of this work was i) feasibility of this method in dogs and ii) identification of potential arrhythmic risk factors that could explain overmortality during obesity. METHOD: Six dogs received a high fat diet (HFD) ad libitum during 20 weeks. A 24 hour EKG recording was realized just before and after 20 weeks of HFD. The following parameters studying QT interval were collected: QT interval lasting from the beginning of the Q wave to the apex (QTa) and to the end of the T wave (QTe), QT intervals plotted against RR intervals and two regression lines were calculated characterized by their slope and intersection with the Y axis, QT dispersion (longest minus shortest QT interval for each RR value) as well as the difference of QT interval between night and day at a fixed RR value considered as a marker of the sympathovagal balance. Our results show that HFD significantly increased body weight, blood pressure, heart rate, left ventricular mass and insulinemia. QT dispersion was increased in a non-significant manner both during day (+35%) and night (16%) for QTa and only during day for QTe (+27%). This increased dispersion of QT was not associated to any increase of QT interval. There was no effect of HFD on QT dynamicity parameter nor on the night-day difference at any RR interval from 300 to 1,300 ms. CONCLUSION: HFD tend increase QT dispersion without any effect on QT interval. These results are compatible with a heterogeneous repolarization probably related to abnormal autonomic nervous system tone. This study could partly explain occurrence of lethal arrhythmias during obesity which might lead to overmortality of obese patients. These results are different for QTa and QTe, but these two parameters are characterizing different type of ventricular cells. This study confirms the feasibility of this method in an experimental model, but results need to be validated in larger groups and in human.

[Early atrial gene regulation of obesity-related arterial hypertension]. / Modifications précoces du transcriptome auriculaire dans l'hypertension artérielle associée à l'obésité chez le chien.

High fat diet (HFD) induces both arterial hypertension and tachycardia in dogs. Changes in heart rate occur early and are in part due to a decrease in the parasympathetic drive to the heart secondary to down-regulation of atrial muscarinic M2 receptors (Pelat et al. Hypertension 1999; 340: 1066-72). These data suggest that HFD is able to modify genic expression at atrial level. Thus, the aim of this work was to perform a systematic study of the genic expression profile in dogs made obese and hypertensive by 9 weeks of HFD. Blood pressure and heart rate were measured by telemetry implanted 15 days before starting regimen in 6 HFD and in 6 control dogs. HFD was the normal canine diet administered to controls but mixed with 300 g of beef fat. At the end of the experience, animals were sacrified and right atria were collected. Gene regulation was assessed in pooled tissue samples from both groups using suppressive substractive hybridization and microarray analysis. Genes with induction or repression rates of at least 20% when compared to controls were sequenced. As previously reported HFD induced a significant increase in body weight, blood pressure and heart rate when compared to controls. The results of SSH experiments led to the identification of 32 genes which are differentially regulated in atria from HFD dogs. Most are genes encoding proteins which have been previously shown to be regulated during various cardiopathies (MMP9, Na/K-ATPase 3...). These changes indicate the existence of early remodeling processes of atrial myocardium secondary to HFD. Other group of genes encodes proteins with no role identified in heart up today (lec-3, ERK-3, TRIP1, nucleophosmin...) or which function remains totally unknown. This work confirms that HFD is associated with early changes in gene expression in atrium. These changes are unlikely to be related to ventricular hypertrophy which is observed only during long-term HFD. Further studies are necessary to demonstrate the role of these modifications in the pathophysiological mechanisms leading to the increase in heart rate in this model of obesity-related arterial hypertension.

Semicarbazide-sensitive amine oxidase substrates stimulate glucose transport and inhibit lipolysis in human adipocytes.

Semicarbazide-sensitive amine oxidases (SSAO) are widely distributed enzymes scavenging biogenic or exogenous amines and generating hydrogen peroxide. We asked whether human adipose tissue could express SSAO. Since hydrogen peroxide exhibits pharmacological insulin-like effects, we also tested whether its endogenous production by SSAO could mimic several insulin effects on adipocytes, such as stimulation of glucose uptake and inhibition of lipolysis. The benzylamine oxidation by human adipose tissue was inhibited by semicarbazide or hydralazine and resistant to pargyline or selegiline. It was due to an SSAO activity localized in adipocyte membranes. A protein of 100-kDa and a 4-kb mRNA corresponding to SSAO were identified in either mammary or abdominal subcutaneous fat depots. In isolated adipocytes, SSAO oxidized similarly benzylamine and methylamine that dose dependently stimulated glucose transport in a semicarbazide-sensitive manner. Antioxidants also inhibited the benzylamine and methylamine effects. Moreover, the ability of diverse substrates to be oxidized by adipocytes was correlated to their effect on glucose transport. Benzylamine and methylamine exerted antilipolytic effects with a maximum attained at 1 mM. These results show that human adipocytes express a membrane-bound SSAO that not only readily oxidizes exogenous amines and generates H(2)O(2), but that also interplays with glucose and lipid metabolism by exerting insulin-like actions. Based on these results and the fact that variations in plasma levels of the soluble form of SSAO have been previously reported in diabetes, we propose that determination of adipocyte SSAO, feasible on subcutaneous microbiopsies, could bring relevant information in pathologies such as obesity or diabetes.

The alpha1-microglobulin/bikunin gene: characterization in mouse and evolution.

The 129Sv mouse gene coding for the alpha1-microglobulin/bikunin precursor has been isolated and characterized. The 11kb long gene contains ten exons, including six 5'-exons coding for alpha1-microglobulin and four 3'-exons encoding bikunin. Exon 7 also codes for the tribasic tetrapeptide RARR which connects the alpha1-microglobulin and bikunin parts. The sixth intron, which separates the alpha1-microglobulin and bikunin encoding parts, was compared in the human, mouse and a fish (plaice) gene. The size of this intron varies considerably, 6.5, 3.3 and 0.1kb in man, mouse and plaice, respectively. In all three genes, this intron contains A/T-rich regions, and retroposon elements are found in the first two genes. This indicates that this sixth intron is an unstable region and a hotspot for recombinational events, supporting the concept that the alpha1-microglobulin and bikunin parts of this gene are assembled from two ancestral genes. Finally, the nonsynonymous nucleotide substitution rate of the gene was determined by comparing coding sequences from ten vertebrate species. The results indicate that the alpha1-microglobulin part of the gene has evolved faster than the bikunin part.

Matrix metalloproteinase gelatinases in sulfur mustard-induced acute airway injury in guinea pigs.

Respiratory tract lesions induced by sulfur mustard (SM), a chemical warfare agent, are characterized by epithelial damage associated with inflammatory cell infiltration. To test the potential role of matrix metalloproteinase gelatinases in these lesions, we evaluated gelatinase activity, albumin content, and total cell count in bronchoalveolar lavage fluid of guinea pigs 24 h after an intratracheal injection of 0.2 mg/kg of SM. The bronchial lavage and alveolar lavage fluids were analyzed separately. The increase in inflammatory cell content of the bronchial lavage fluid, mainly macrophages, observed in SM-intoxicated guinea pigs was accompanied by an increase in albumin and in 92-kDa gelatinase activity. There was a significant correlation between albumin content and 92-kDa gelatinase activity (r = 0.67) and between 92-kDa gelatinase and the number of macrophages. Immunohistochemistry performed on tracheal sections showed the presence of 92-kDa gelatinase at the site of intraepithelial cleavages. Zymography analysis of culture medium conditioned by guinea pig tracheal epithelial cells demonstrated that these cells produced in vitro 92-kDa gelatinase on stimulation. Culture of human bronchial epithelial cells obtained by the explant technique showed a marked increase in 92-kDa gelatinase after exposure to 5 x 10(-5) M SM that reinforced the relevance of our animal results to human exposure to SM. These results suggest that in SM respiratory intoxication, 92-kDa gelatinase of both inflammatory and epithelial cell origins could be involved in epithelial cell detachment.

Testis expression of hormone-sensitive lipase is conferred by a specific promoter that contains four regions binding testicular nuclear proteins.

The testicular isoform of hormone-sensitive lipase (HSLtes) is encoded by a testis-specific exon and 9 exons common to the testis and adipocyte isoforms. In mouse, HSLtes mRNA appeared during spermiogenesis in round spermatids. Two constructs containing 1.4 and 0.5 kilobase pairs (kb) of the human HSLtes gene 5'-flanking region cloned upstream of the chloramphenicol acetyltransferase gene were microinjected into mouse oocytes. Analyses of enzyme activity in male and female transgenic mice showed that 0.5 kb of the HSLtes promoter was sufficient to direct expression only in testis. Cell transfection experiments showed that CREMtau, a testis-specific transcriptional activator, does not transactivate the HSLtes promoter. Using gel retardation assays, four testis-specific binding regions (TSBR) were identified using testis and liver nuclear extracts. The testis-specific protein binding on TSBR4 was selectively competed by a probe containing a SRY/Sox protein DNA recognition site. Sox5 and Sox6 which are expressed in post-meiotic germ cells bound TSBR4. Mutation of the AACAAAG motif in TSBR4 abolished the binding. Moreover, binding of the high mobility group domain of Sox5 induced a bend within TSBR4. Together, our results showed that 0.5 kb of the human HSLtes promoter bind Sox proteins and contain cis-acting elements essential for the testis specificity of HSL.

An array of binding sites for hepatocyte nuclear factor 4 of high and low affinities modulates the liver-specific enhancer for the human alpha1-microglobulin/bikunin precursor.

Alpha1-Microglobulin and bikunin are two plasma glycoproteins encoded by a gene for alpha1-microglobulin/bikunin precursor (AMBP). The strict liver-specific transcription of the AMBP gene is controlled by an elaborate and remote enhancer made of six clustered boxes numbered 1 to 6 (core enhancer) that are binding sites for the hepatocyte-enriched nuclear factors HNF-1, HNF-4, HNF-3, HNF-1, HNF-3 and HNF-4 respectively. Three further boxes, 7 to 9, have now been found in the enhancer area in a position 5' of box 2, 5' of box 1 and 3' of box 6, respectively. Electrophoretic mobility-shift assays with nuclear extracts from the HepG2 hepatoma cell line demonstrated that boxes 7 and 8 are both functional HNF-4-binding sites of high and low affinity respectively, whereas no binding capacity of box 9 was detected by this method. Transfection of HepG2 and Chinese hamster ovary cells with chloramphenicol acetyltransferase constructs harbouring the core or extended AMBP enhancer with wild-type or mutated boxes and co-transfection with expression plasmids for a wild-type or defective HNF-4 identified box 7 as an essential element for the basal activity of this enhancer. The response of boxes 7 and 8 varies with the level of HNF-4 in cells. Box 9 exhibits a repressor activity that can be detected when box 8 is ablated. In vivo this corresponds to conditions of low box 8 occupancy when the intracellular level of HNF-4 is limited. These results reinforce the view that the AMBP enhancer is a quite elaborate and unusual example of a modular enhancer whose activity is fine-tuned by the level of cognate nuclear factors in the cell.

Down-regulation of negative acute-phase response genes by hypotonic stress in HepG2 hepatoma cells.

An increased hepatocellular hydration state (HS) that can be induced by hypotonic stress or a high glutamine uptake modulates the transcription of given genes in liver. This could be important in the acute phase (AP) of a systemic inflammation where both HS and glutamine uptake transiently increase in liver. In HepG2 hepatoma cells cultured in conditions of hypotonic stress or a high extracellular glutamine availability, a specifically decreased expression of two human mRNAs, namely those of alphal-microglobulin/bikunin precursor (AMBP) and alpha2-HS-glycoprotein, that are also down-regulated in liver by AP, could be seen. A functional analysis of the AMBP promoter indicated that this hypotonic stress-induced down-regulation takes place at a transcriptional level. In these experiments, the mRNA level and transcription of the glyceraldehyde-3-phosphate dehydrogenase gene that are known to be unmodified in AP did not exhibit any change. Given that hypotonic stress also upregulates the transcription of a liver gene that is also upregulated in AP [Meisse et al. (1998) FEBS Lett. 422, 3463481, the AP-associated increase in hepatocellular HS now appears to participate in the transcriptional control of both sets of genes that are up- or down-regulated in AP.

The inter-alpha-inhibitor family: from structure to regulation.

Inter-alpha-inhibitor (IalphaI) and related molecules, collectively referred to as the IalphaI family, are a group of plasma protease inhibitors. They display attractive features such as precursor polypeptides that give rise to mature chains with quite distinct fates and functions, and inter-chain glycosaminoglycan bonds within the various molecules. The discovery of an ever growing number of such molecules has raised pertinent questions about their pathophysiological functions. The knowledge of this family has long been structure-oriented, whereas the structure/function and structure/regulation relationships of the family members and their genes have been largely ignored. These relationships are now being elucidated in events such as gene transcription, precursor processing, changes in plasma protein levels in health and disease and binding capacities that involve hyaluronan as well as other plasma proteins as ligands. This review presents some recent progress made in these fields that paves the way for an understanding of the functions of IalphaI family members in vivo. Finally, given the wealth of heterogeneous, complicated and sometimes contradictory nomenclatures and acronyms currently in use for this family, a new, uniform, nomenclature is proposed for IalphaI family genes, precursor polypeptides and assembled proteins.

Double-strand breaks at the target locus stimulate gene targeting in embryonic stem cells.

Double-strand breaks (DSBs) are recombinogenic lesions in chromosomal DNA in yeast, Drosophila and Caenorhabditis elegans. Recent studies in mammalian cells utilizing the I-Scel endonuclease have demonstrated that in some immortalized cell lines DSBs in chromosomal DNA are also recombinogenic. We have now tested embryonic stem (ES) cells, a non-transformed mouse cell line frequently used in gene targeting studies. We find that a DSB introduced by I-Scel stimulates gene targeting at a selectable neo locus at least 50-fold. The enhanced level of targeting is achieved by transient expression of the I-Scel endonuclease. In 97% of targeted clones a single base pair polymorphism in the transfected homologous fragment was incorporated into the target locus. Analysis of the targeted locus demonstrated that most of the homologous recombination events were 'two-sided', in contrast to previous studies in 3T3 cells in which 'one-sided' homologous events predominated. Thus ES cells may be more faithful in incorporating homologous fragments into their genome than other cells in culture.

Hierarchy and positive/negative interplays of the hepatocyte nuclear factors HNF-1, -3 and -4 in the liver-specific enhancer for the human alpha-1-microglobulin/bikunin precursor.

Alpha-1-microglobulin and bikunin are two plasma glycoproteins encoded by an alpha-1-microglobulin/bikunin precursor (AMBP) gene. The strict liver-specific expression of the AMBP gene is controlled by a potent enhancer made of six clustered boxes numbered 1-6 that have been reported to be proven or potential binding sites for the hepatocyte-enriched nuclear factors HNF-1, -4, -3, -1, -3, -4, respectively. In the present study, electromobility shift assays of wild-type or mutated probes demonstrated that the boxes 1-5 have a binding capacity for their cognate HNF protein. Box 5 is also a target for another, as yet unidentified, factor. A functional analysis of the wild-type or mutated enhancer, driving its homologous promoter and a reporter CAT gene in the HepG2 hepatoma cell line, demonstrated that all six boxes participate in the enhancer activity, with the primary influence of box 4 (HNF-1) and box 2 (HNF-4). A similar analysis in the HNF-free CHO cell line co-transfected with one or several HNF factors further demonstrated various interplays between boxes: box 3 (HNF-3 alpha and beta) has a negative influence over the major HNF-4 box 2 as well as a positive influence over the major HNF-1 box 4.

Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease.

To maintain genomic integrity, double-strand breaks (DSBs) in chromosomal DNA must be repaired. In mammalian systems, the analysis of the repair of chromosomal DSBs has been limited by the inability to introduce well-defined DSBs in genomic DNA. In this study, we created specific DSBs in mouse chromosomes for the first time, using an expression system for a rare-cutting endonuclease, I-SceI. A genetic assay has been devised to monitor the repair of DSBs, whereby cleavage sites for I-SceI have been integrated into the mouse genome in two tandem neomycin phosphotransferase genes. We find that cleavage of the I-SceI sites is very efficient, with at least 12% of stably transfected cells having at least one cleavage event and, of these, more than 70% have undergone cleavage at both I-SceI sites. Cleavage of both sites in a fraction of clones deletes 3.8 kb of intervening chromosomal sequences. We find that the DSBs are repaired by both homologous and nonhomologous mechanisms. Nonhomologous repair events frequently result in small deletions after rejoining of the two DNA ends. Some of these appear to occur by simple blunt-ended ligation, whereas several others may occur through annealing of short regions of terminal homology. The DSBs are apparently recombinogenic, stimulating gene targeting of a homologous fragment by more than 2 orders of magnitude. Whereas gene-targeted clones are nearly undetectable without endonuclease expression, they represent approximately 10% of cells transfected with the I-SceI expression vector. Gene targeted clones are of two major types, those that occur by two-sided homologous recombination with the homologous fragment and those that occur by one-sided homologous recombination. Our results are expected to impact a number of areas in the study of mammalian genome dynamics, including the analysis of the repair of DSBs and homologous recombination and, potentially, molecular genetic analyses of mammalian genomes.

Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells.

Double-strand breaks introduced into DNA in vivo have been shown to enhance homologous recombination in a variety of chromosomal and extrachromosomal loci in Saccharomyces cerevisiae. To introduce double-strand breaks in DNA at defined locations in mammalian cells, we have constructed a mammalian expression vector for a modified form of I-Sce I, a yeast mitochondrial intron-encoded endonuclease with an 18-bp recognition sequence. Expression of the modified I-Sce I endonuclease in COS1 cells results in cleavage of model recombination substrates and enhanced extrachromosomal recombination, as assayed by chloramphenicol acetyltransferase activity and Southern blot analysis. Constitutive expression of the endonuclease in mouse 3T3 cells is not lethal, possibly due to either the lack of I-Sce I sites in the genome or sufficient repair of them. Expression of an endonuclease with such a long recognition sequence will provide a powerful approach to studying a number of molecular processes in mammalian cells, including homologous recombination.

Human inter-alpha-inhibitor family in inflammation: simultaneous synthesis of positive and negative acute-phase proteins.

The inter-alpha-inhibitor (I alpha I) family encompasses four plasma proteins, namely free bikunin as well as I alpha I, pre-alpha-inhibitor (P alpha I) and inter-alpha-like inhibitor (I alpha LI). Each of the last three proteins is a distinct assembly of one bikunin chain with one or more unique heavy (H) chains designated H1, H2 and H3. The three H chains and the bikunin chain are encoded by four distinct mRNAs. These molecules and chains, as well as the corresponding mRNAs, were quantified in sera and liver biopsies from a series of patients with or without mild or severe acute infection. The decrease or increase observed for a given molecule or chain in the serum was in agreement with a similar change in the corresponding liver mRNA. In acute inflammation the H2 and bikunin chains are down-regulated and the relevant molecules (I alpha I, I alpha LI) behave as negative acute-phase proteins, whereas the H3 chain is up-regulated and the corresponding P alpha I molecule is a positive acute-phase protein. Also, P alpha I displays a higher-than-usual M(r); this is probably due to ligand binding. The H1 gene does not seem to be affected by the inflammatory condition. The quantitative changes in RNA levels seen in vivo were confirmed in vitro in the human hepatoma Hep3B cell line prior to or after induction with the acute-phase mediators interleukin-1 and/or -6. These results provide the first example in humans of positive and negative acute-phase proteins that are encoded by evolutionary related genes.